- Email: [email protected]
New cytotoxic steroidal saponins from Cestrum parqui
Phytochemistry Letters 22 (2017) 167–173
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
New cytotoxic steroidal saponins from Cestrum parqui a
b
a
MARK a
b
Radwa R. Mosad , Mohamed H. Ali , Magda T. Ibrahim , Hala M. Shaaban , Marwan Emara , ⁎ Amir E. Wahbac, a b c
Department of Pharmacognosy, Faculty of Pharmacy For Girls, Al-Azhar University, Egypt Center of Aging and Associated Diseases, Zewail City of Science and Technology, Egypt Chemistry Department, Faculty of Science, Damietta University, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords: Cestrum parqui Steroidal saponins Spirostanol Furostanol Cytotoxicity Digalogenin
Two new steroidal saponins, 25(R)-3β [(O-β-D-glucopyranosyl-(1 → 3)-β-D-glucopyranosyl-(1 → 2)-O-[β-D-xylopyranosyl-(1 → 3)-O-β-D-glucopyranosyl-(1 → 4)-β-D-galactopyranosyl)oxy]-5α, 15β, 22R, 25R-spirostan3,15-diol (1, named parquispiroside) and 25R-26-[(β-D-glucopyranosyl)Oxy]-(3β [(O-β-D-glucopyranosyl(1 → 3)-β-D-glucopyranosyl-(1 → 2)-O-[β-D-xylopyranosyl-(1 → 3)-O-β-D-glucopyranosyl-(1 → 4)-β-D-galactopyranosyl)oxy], 5α, 15β, 22R, 25R)-furostane-3,15,22-triol (2, named parquifuroside), along with the known saponins, capsicoside D (3) and 22-OMe-capsicoside D (4) and the known glycoside, benzyl primeveroside (5), were isolated from the leaves of Cestrum parqui. The structures of these compounds were elucidated by careful analysis of 1D and 2D NMR spectra and ESIMS data. Parquispiroside (1) exhibited moderate inhibition of Hela, HepG2, U87, and MCF7 cell lines with IC50 values in the range of 3.3–14.1 μM.
1. Introduction Saponins are structurally complex glycosides composed of a nonpolar steroid, or triterpene, modified with one or more highly polar sugar moieties. The resulting amphiphilic physical properties of saponins provide for unique interactions with living cells. Several biological activities have been reported for saponins including: anticancer, antiviral, hypoglycemic, anti-inflammatory, heamolytic, and antifungal (Sparg et al., 2004). Specifically, saponins have the ability to bind the phospholipid bilayer and cholesterol to form pores in the cell membrane and cause cell lysis (Lorent et al., 2014). Several saponins have been shown to induce cell death by multiple mechanisms including apoptosis, autophagy, and necrosis (Lorent et al., 2014), suggesting a potential to evade drug-resistance. However, cancer treatment with saponins suffers the limitation of a narrow therapeutic window due to potential toxicity. The emerging field of nanoformulation could help to increase the therapeutic window and hence open the door for the possibility of using pure and/or mixtures of saponins in clinical applications (Rejinold et al., 2011; Hu et al., 2010). As a part of our program to identify anticancer drug leads from the Egyptian environment, we investigated the methanolic extract of the leaves of Cestrum parqui, a shrub commonly known as the green cestrum. While many plants of the genus Cestrum, the second largest genus of the Solanaceae family (Judd et al., 1999), have been explored extensively, natural products from Cestrum parqui occur in little reports to ⁎
date. The plant is commonly known for its toxicity to cattle (McLennan and Kelly, 1984). Saponins are the major group of natural products isolated from Cestrum parqui (Baqui et al., 2001; D'Abrosca et al., 2004; Silva et al., 1962). In this study, we report the isolation, structure elucidation and anticancer evaluation of two new steroidal saponins along with three known compounds from the leaves of Cestrum parqui. 2. Material and methods 2.1. General experimental procedure Nuclear Magnetic Resonance spectra (1D and 2D) were recorded with a Bruker Ascend™ spectrometer (Bruker Daltonics, Bremen, Germany) at 400 MHz for proton, and 100 MHz for carbon. The data obtained were processed using ACD-NMR processor software ver. 12.01. The solvents used were CD3OD and DMSO d6. Chemical shift data was reported in parts per million (ppm) on the δ scale; coupling constants (J) were expressed in Hz. LC–MS/MS was carried out using a Waters TQ detector, Acquity ultra performance LC binary solvent manager and auto sampler operated by Mass Lynx software. Pre-coated silica gel plates (TLC) F254 (20 × 20 cm) were used for analytical separation, using ascending technique and suitable solvent systems with visualization using p-anisaldehyde/H2SO4 and vanillin solution. Normal phase chromatography was carried out using silica gel G60-230 (Merck, Darmstadt, Germany) packed by the wet method in the stated solvent.
Corresponding author. E-mail address: [email protected] (A.E. Wahba).
http://dx.doi.org/10.1016/j.phytol.2017.09.022 Received 20 June 2017; Received in revised form 24 September 2017; Accepted 27 September 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 22 (2017) 167–173
R.R. Mosad et al.
Sephadex LH-20 column chromatography was carried out using sephadex (SIGMA-ALDRICH, Missouri, USA). Reversed phase chromatography was performed using phase-bonded octadecylsilyl-silica gel (RPC18, BAKERBOND® Octadecyl, C18) 40 μm, Prep LC packing (Phillipsburg, NJ, USA).
Table 1 NMR spectroscopic data for parquispiroside(1) and parquifuroside (2). Position
Parquispiroside (1) (DMSO, d6) δC,
2.2. Plant materials Fresh leaves of Cestrum parqui L’Hér were collected in November 2014 from plants cultivated in El-Kanater El-Khairia gardens and Ahmed Alaa’s garden in Shebeen Elkom, Menoufia, Egypt. The plant was identified and confirmed by Prof. Abd El-Hamid Khedr, Department of Botany, Faculty of Science, Damietta University, Damietta, Egypt. A voucher specimen was kept at the Department of Pharmacognosy, Faculty of Pharmacy for Girls, AlAzhar University and was given the reference number (CP01). 2.3. Extraction and isolation The air dried, powdered leaves (1 kg) were extracted by maceration with cold distilled CH3OH (10 L) for four days. The methanolic extract was concentrated under reduced pressure to constant weight (≈150 g). The dried methanolic residue was dissolved in the least volume of MeOH, loaded onto 100 g silica gel and chromatographed over a silica gel column (150 × 5 cm, i.d., 500 g) elution started using 100% petroleum ether, then increasing polarity gradually using petroleum ether: methylene chloride, (wa EtOAc)1 till 100% CH3OH (10% increment) and then the column was washed with n-butanol. Fractions of 100 mL each were collected, concentrated, and monitored by TLC with an appropriate solvent system. Five pools were collected. Pool 4 (fractions eluted with 100% wa EtOAc up to 100% MeOH were combined, 22.1 g) was chromatographed over silica gel column (100 × 2.5 cm i.d., 160 g) and eluted with pet. ether: wa EtOAc mixtures with gradual increase of polarity (10% increments) till 100% wa EtOAc then wa EtOAc: CH3OH up to 100% CH3OH. 800 fractions were collected (20 mL each) and monitored by TLC. Fractions 701–738 were combined and compound 1 was precipitated out as a pure white amorphous solid. Fractions 750–800 were combined and further purified over RP-C18 column with an isocratic solvent system (MeOH:H2O 1:9, 10 mL fraction size). Fractions 5–20 were combined and purified over a preparative TLC plate with a mixture of 12:8:8:2 (wa EtOAc: DCM: MeOH: H2O) as the eluting system to give compound 5. Pool 5 (n-butanol fraction, 9 g) was fractionated over silica gel with wa EtOAc to MeOH step gradient to produce four fractions. Fraction 4 (100% MeOH, 1.2 g) was further purified over a RP-C18 column using the isocratic solvent system, MeOH:H2O 3:7 that gave pure compounds 2–4.
type
1
36.2, CH2
2
29.6, CH2
3 4
76.2, CH 33.8, CH2
5 6
44.0, CH 28.1, CH2
7
30.3, CH2
8 9 10 11
30.6, 53.7, 35.2, 20.6,
CH CH C CH2
δH (J Hz)
δC,type
δH (J Hz)
1⋅62 0.92 1⋅74 1.38 3.54 1.61 1.17 1.01 1.60 1.24 1.76 1.41 1.76 0.68 – 1.43 1.28
38.0, CH2
1.75 (m) 1.06 (m) 1.7 (m) 1.4 (m) 3.69 (m) 1.57 (m) 1.33 (m) 1.15 (m) 1.92 (m) 1.59 (m) 2.00 (m) 0.99 (m) 1.8 (m) 0.86 (m) –
(m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m)
12
2.3.1. Parquispiroside (1); 25(R)-3β [(O-β-D-glucopyranosyl-(1 → 3)-β-Dglucopyranosyl-(1 → 2)-O-[β-D-xylopyranosyl-(1 → 3)-O-β-Dglucopyranosyl-(1 → 4)-β-D-galactopyranosyl)oxy]-5α, 15β, 22R, 25Rspirostan-3,15-diol Amorphous white solid (MeOH), 5.7 mg; 1H NMR (DMSO, d6, 400 MHz) and 13C NMR (DMSO, d6, 400 MHz) see Table 1; ESI–MS (+ve) m/z 1242 [M+K]+ 2.3.2. Parquifuroside (2): 25R-26-[(β-D-glucopyranosyl)Oxy]-(3β [(O-βD-glucopyranosyl-(1 → 3)-β-D-glucopyranosyl-(1 → 2)-O-[β-Dxylopyranosyl-(1 → 3)-O-β-D-glucopyranosyl-(1 → 4)-β-Dgalactopyranosyl)oxy], 5α, 15β, 22R, 25R)-furostane-3,15,22-triol Amorphous white solid (MeOH), 7.0 mg; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 400 MHz) see Table 1; ESI–MS (+ve) m/z 1432 [M+K]+ 1 Commercial ethyl acetate was mixed with 3% (v/v) water and 1% (v/v) acetic acid and shake well until clear solution was observed.
Parquifuroside (2) (CD3OD)
29.7, CH2 79.3, CH 28.5, CH2 44.8, CH 29.0, CH2 31.5, CH2 35.0, 54.9, 35.9, 23.8,
CH CH C CH2
36.3, CH2
13 14 15 16 17 18 19 20 21 22 23
40.4, C 58.9, CH 68.6, CH 82.3, CH 60.6, CH 18.2, CH3 11.8, CH3 41.7, CH 14.0, CH3 109.1, C 31.1, CH2
– 0.93 3.93 4.13 1.80 0.95 0.80 1.83 0.88 – 1.86
24
28.8, CH2
25 26
29.5, CH 65.6, CH2
1.77 1.40 1.53 3.43 3.10 0.75
27
16.9, CH3
1 2 3 4 5 6
Gal-I 100.7, CH 71.2, CH 73.3, CH 78.4, CH 76.2, CH 59.3, CH2
1 2 3 4 5 6
Glu-I 102.8, CH 78.3, CH 84.6, CH 68.5, CH 76.2, CH 60.5, CH2
1 2 3 4 5 6
Glu-II 101.1, CH 72.7, CH 86.9, CH 67.9, CH 76.2, CH 60.8, CH2
1 2 3 4 5 6
Glu-III 103.6, CH 73.6, CH 75.8, CH 69.9, CH 76.6, CH 61.0, CH2
4.22 3.24 3.35 3.78 3.01 3.72 3.40
(m) (dd, 4.9, 3.7) (dd, 5.38, 5.38) (m) (s) (s) (m) (d, 6.0) (m), 1.61 (m) (m) (m) (m) (m) (m) (d, 6.0) (d, 7.6) (m) (m) (m) (m) (m) (m)
4.45 (d, 7.6) 3.61 (m) 3.62 (m) 3.13 (m) 3.12 (m) 3.7 (m) 3.38 (m) 4.87 (d, 7.6) 3.23 (m) 3.37 (m) 3.26 (m) 3.10 (m) 3.4 (m) 3.68 (m) 4.32 3.06 3.18 3.10 3.27 3.67 3.34
(d, 7.8) (m) (m) (m) (m) (m) (m)
44.7, C 59.5, CH 79.8, CH 84.0, CH 61.3, CH 41.8, CH3 14.2, CH3 13.1, CH 12.5, CH3 115.2, C 35.2, CH2 30.3, CH2 35.0, CH 76.0, CH2 17.2, CH3 Gal-I 102.6, CH 73.0, CH 75.2, CH 79.8, CH 75.5, CH 61.0, CH2 Glu-I 104.5, CH 80.7, CH 87.5, CH 70.4, CH 77.3, CH 61.0, CH2 Glu-II 103.6, CH 75.2, CH 87.4, CH 70.0, CH 78.0, CH 62.5, CH2 Glu-III 105.0, CH 75.5, CH 77.8, CH 71.5, CH 77.0, CH 60.0, CH2
1.8 (m) 1.72 (m) – 0.97 (m) 4.12 (dd, 4.9, 3.6) 4.43 (dd, 5.1, 3.9) 2.17 (dd, 11.3, 9) 2.10 (m) 1.05 (d, 6.6) 1.03 (s) 0.92 (s) – 1.85 (m) 1.74 (m) 1.71 (m) 1.1 (m) 1.78 (m) 3.78 (m) 3.41 (m) 0.97 (d, 6.4) 4.40 3.64 3.54 4.02 3.51 3.90 3.68
(d, 7.7) (m) (m) (m) (m) (m) (m)
4.62 3.80 3.74 3.32 3.39 3.90 3.68
(d, 7.6) (m) (m) (m) (m) (m) (m)
5.0 (d, 7.7) 3.43 (m) 3.60 (m) 3.48 (m) 3.74 (m) 3.83 (m) 3.65 (m) 4.60 3.32 3.37 3.66 3.34 3.87 3.69
(d, 7.7) (m) (m) (m) (m) (m) (m)
(continued on next page)
168
Phytochemistry Letters 22 (2017) 167–173
R.R. Mosad et al.
prepared in DMSO (Sigma Aldrich, USA). Working concentrations contained less than 0.15% (v/v) DMSO and the same amount of DMSO was added to the control group as the experimental group. Cisplatin positive control was used at the different concentrations known to induce cytotoxic effect in different cell lines [HeLa (40 μM), HepG2 (20 μM), U-87 MG (25 μM), and MCF7 (10 μM)]. All treated and untreated cultures were incubated for 48 h followed by addition of CCK-8 solution (10 μL/well), incubation for two additional hours, and absorbance measurement at 450 nm using microplate reader (FLUOstar Omega, BMG LABTECH GmbH, Germany). The number of living cells is directly proportional to the amount of formazan dye generated by dehydrogenases in these cells. The percentage of viable cells was calculated according to the following formula:
Table 1 (continued) Position
Parquispiroside (1) (DMSO, d6) δC,
type
1 2 3 4 5
Xyl 103.0, CH 75.7, CH 76.2, CH 69.1, CH 65.9, CH2
1 2 3 4 5 6
– – – – – –
δH (J Hz)
4.51 3.10 3.22 3.34 3.44 3.24
(d, 7.6) (m) (m) (m) (m) (m)
– – – – – –
Parquifuroside (2) (CD3OD) δC,type Xyl 104.8, CH 74.0, CH 77.9, CH 69.3, CH 67.0, CH2
104.6, CH 75.0, CH 77.6, CH 71.3, CH 76.6, CH 61.2, CH2
δH (J Hz)
4.65 3.27 3.38 3.32 3.95 3.27
(d, 7.7) (m) (m) (m) (m) (m)
Glu-IV 4.30 (d, 7.7) 3.2 (m) 3.37 (m) 3.31 (m) 3.35 (m) 3.95 (m) 3.68 (m)
Cell Viability (%) =
A sample − Ablank A control − Ablank
× 100
2.6. Statistical analysis Data from three biological replicate experiments with three technical replicate each were expressed as mean ± SE. Statistical analysis was carried out using SigmaPlot 13 (Systat software, Inc, USA). The IC50 was calculated using Microcal Origin software program (Microcal software Inc., USA).
2.3.3. Capsicoside D (3) Amorphous white solid (MeOH), 3.0 mg; 1H NMR (CD3OD, 400 MHz): δ 5.0 (1H, d, J = 7.7), 4.65 (1H, d, J = 7.7), 4.62 (1H, d, J = 7.6), 4.60 (1H, d, J = 7.7), 4.40 (1H, d, J = 7.7), 4.30 (1H, d, J = 7.7), 3.78 (1H, m), 3.69 (1H, m), 3.41 (1H, m), 1.05 (3H, d, J = 6.6), 1.03 (3H, s),0.97 (3H, d, J = 6.4), 0.92 (3H, s).
3. Results and discussion
2.3.4. 22-OMe capsicoside D (4) Amorphous white solid (MeOH), 2.3 mg; 1H NMR (CD3OD, 400 MHz): δ 5.0 (1H, d, J = 7.7), 4.65 (1H, d, J = 7.7), 4.62 (1H, d, J = 7.6), 4.60 (1H, d, J = 7.7), 4.40 (1H, d, J = 7.7), 4.30 (1H, d, J = 7.7), 3.78 (1H, m), 3.69 (1H, m), 3.41 (1H, m), 3.23 (3H, s), 1.05 (3H, d, J = 6.6), 1.03 (3H, s),0.97 (3H, d, J = 6.4), 0.92 (3H, s).
Compound 1, parquispiroside, was obtained as a white solid. A molecular formula of C56H92O28 was proposed for 1 from the ESI–MS spectrum, which contained a major ion at m/z 1242 [M+K]+ and was supported by the presence of 56 carbon resonances in the 13C NMR spectrum. The 1H NMR revealed the four methyl resonances typical of the steroid class, two of which were secondary at δH 0.75 (d, J = 6.1 Hz) and δH 0.88 (d, J = 6.1 Hz) while the remaining two were quaternary at δΗ 0.81 and δΗ 0.95. Five anomeric protons were also observed at δΗ 4.22 (d, J = 7.3 Hz), 4.32 (d, J = 7.8 Hz), 4.45 (d, J = 7.0 Hz), 4.51 (d, J = 7.6 Hz), and 4.86 (d, J = 7.6 Hz). The 13C NMR and DEPT135 spectra of 1 confirmed the steroidal glycoside nature of the compound in which 29 out of the 56 resonances were assigned for the penta-saccharide fragment. The remaining carbon resonances were assigned to the aglycone, which included the characteristic acetal carbon resonance for a spirostanol at δC 109.1 (C-22) (Agrawal et al., 1995) and three oxygenated methines at δC 82.3, 76.2 and 68.6. The downfield shifts of the resonances for C-15 and the C-16 methine to δC 68.6 and δC 82.8, respectively, suggested a hydroxylation at C-15. The HMBC and 1H-1H–COSY correlations of the methine proton H-16 at δH 4.13 (d, J = 5.4, 5.3 Hz) with carbon resonance at δC 68.6 and H-15 at δΗ 3.93 (m) confirmed the hydroxylation at C-15. The relative configuration of the newly introduced hyroxy group at C-15 is assigned as β orientation based on the strong NOESY correlations between H-15 and Me-21 (δΗ 0.88, d, J = 6.1 Hz), H-16 (δΗ 4.13, dd, J = 5.4, 5.3 Hz), H-17 (δΗ 1.83, m) and H-14 (δΗ 0.93, m). Mutual NOESY correlations between protons attached to rings D and E (H-14, H-16 and H-17) of the steroidal core confirmed the D/E cis junction. The assignment of the F ring was based on the HMBC correlations between the Me-27 protons (δH/C 0.75 (d, J = 6.1 Hz)/16.9) and the oxygenated methylene C-26 (δC 65.6), the C-25 methine (δC 29.5) and C-24 (δC 28.8). The absolute configuration of C-25 is assigned as R based on the chemical shift of Me-27 at δΗ⁄C 0.75/16.9 along with the downfield chemical shifts of C-24 (δC 28.8), C-25 (δC 29.5) and C-26 (δC 65.6) (Agrawal, 2003). The common R-configuration at C-22 is assigned based on the comparison of the chemical shifts of the F ring carbon resonances with the published, yet more rare S-configuration (Hayes et al., 2008). The NOESY correlation between H-5 (δH 1.01 (m)) and H-3 (δH 3.54 (m)) (Fig. 3) along with the carbon chemical shifts of C-5 (δC 44.72), C-
2.3.5. Benzyl primeveroside (5) Amorphous white solid (MeOH), 8.2 mg; 1H NMR (CD3OD, 400 MHz): δ 7.50–7.25 (5H, m), 4.92 (1Η, d, J = 12.0), 4.66 (1H, d, J = 12.0), 4.36 (1H, d, J = 7.6), 4.34 (1H, d, J = 7.6), 4.10 (1H, dd, J = 11.2, 2.1), 3.85 (1H, dd, J = 12.5, 3.4), 3.80 (1H, dd, J = 11.5, 5.9), 3.68 (1H, dd, J = 6.2, 3.2); 13C NMR (CD3OD, 100 MHz): 139.3, 129.4, 129.3, 128.8, 105.4, 103.6, 78.1, 77.2, 75.3, 74.4, 72.6, 72.1, 71.9, 69.7, 69.6, 66.8; ESI–MS (+ve) m/z 425 [M+Na]+ 2.4. Cell lines and in vitro culture conditions Four human cell lines of different cancer origin [HeLa Cells (cervical adenocarcinoma, CCL-2, ATCC, USA), U-87 MG (Glioblastoma, TB-14, ATCC), Hep G2 (HB-8065, ATCC), and MCF7 (Breast adenocarcinoma, HTB-22, ATCC)] were used. HeLa and HepG2 were cultured in RPMI 1640 (12-702F, Lonza, Verviers, Belgium) media, while U-87 MG and MCF7 were cultured in DMEM/F12 (BE12-719F, Lonza) and DMEM (BE12-604F, Lonza) media, respectively. All cells were maintained as monolayer cultures supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, USA) and L-glutamine (BE12-604F, Lonza) in a humidified atmosphere of 5% CO2 in air at 37 °C. No antibiotics or antimycotics were added. 2.5. Cell proliferation assay and cytotoxicity assay The cell proliferation assay and cytotoxicity assays was carried out using Cell counting kit-8 (CCK-8, Code: CK04, Tokyo, Japan). Cells were cultured in a flat bottom 96-microwell plate (4 × 103 cells/100 μL culture media/well) and incubated overnight. Cell lines were treated with the purified compounds at different concentrations (1 μM, 5 μM, 10 μM, 25 μM, and 50 μM) and compared with untreated cell line negative controls. Stock concentrations of the pure compounds were 169
Phytochemistry Letters 22 (2017) 167–173
R.R. Mosad et al.
Fig. 1. Structures of the isolated compounds from Cestrum parqui.
with the anomeric proton at δΗ⁄C 4.22(d, J = 7.34 Hz)/100.7 of the galactose unit. The strong NOESY correlations between H-1′ (δΗ 4.22) and H-3′ (δΗ 3.35) and H-3′ (δΗ 3.35) and H-4′ (δΗ 3.78) confirmed that a galactose unit is attached to the C-3 of the aglycone with a glycosylation at C-4′ (δc 78.4). H-4′ at δH/C (3.78, m)/78.4 of Gal showed HMBC and NOESY correlations with the anomeric carbon at δC/H 102.8/4.45 (d, J = 7.0 Hz) which is assigned to an inner glucose with glycosylation at C-2′′ (δC/H 78.3/3.61 (m)) and C-3′′ (δC/H 84.6/3.62 (m)). The characteristic downfield oxygenated methylene carbon at δC 65.9 for a pentose unit, in addition to the 1H-1H-COSY, HMBC and NOESY correlations of the anomeric proton δΗ⁄C 4.51(d, J = 7.6 Hz)/ 103.0 confirmed a terminal xylose unit to be attached to C-3′′ of GluI. Another hexose unit is attached to C-2′′ of GluI based on the HMBC correlation between H-2′′ with the anomeric carbon δC/H 101.1/4.86 (d, J = 7.6 Hz). Interestingly, this unit was reported to be a galactose in digalonin (Tschesche and Wulff, 1963). However, careful analysis of NOESY correlations of H-3′′′′ (δH/C 3.37 (m)/86.9) revealed strong NOESY correlations with the anomeric proton H-1′′′′ at δΗ 4.86 (d, J = 7.6 Hz) and C2′′′′-OH (δΗ 5.35, d, J = 5.4 Hz) and missing the key NOESY correlation with H-4′′′′ at δΗ 3.25 (m). This suggests that the hydroxy group at C-4′′′′ is at the β-orientation and thus a glucose unit is present (GluII). A terminal glucose unit is attached to C-3′′′′ of GluII in which H-3′′′′ showed an 1,3-HMBC correlations with the anomeric C1′′′′′ of the terminal glucose at δC/H 103.6/4.32. Therefore, analysis of the data in combination with literature comparison confirmed the
7 (δC 29.5) and C-19 (δC 11.8), which they absorbed similarly to the published chemical shifts of trans A/B saponins, suggest the trans fusion between A and B rings (Agrawal et al., 1995). Based on these NMR analyses, the aglycone was identified as (3β, 5α, 15β, 22R, 25R)spirostan-3,15-diol. This aglycone has been reported previously in 1963 (Tschesche and Wulff, 1963), named digalogenin, without NMR data. Thus, we report the first complete NMR assignment of 3-glycosylated digalogenin in DMSO, d6. The 13C NMR of 1 revealed the presence of 5 anomeric carbons at δC 100.7 (Gal), 102.8 (GluI), 103.8 (Xyl), 101.1 (GluII), and 103.6 (GluIII) in which they were correlated to δΗ 4.22 (d, J = 7.34 Hz), 4.45 (d, J = 7.0 Hz), 4.51 (d, J = 7.6 Hz), 4.86 (d, J = 7.6 Hz) and 4.32 (d, J = 7.8 Hz) respectively in the 1H NMR spectra of 1. The large J values (∼7 Hz) of all the anomeric proton resonances suggested the β-configuration of the five anomeric carbons. Unfortunately, there is no NMR data reported for similar saponins dissolved in deuterated DMSO to use for comparison. Thus, all correlations were carefully analyzed in order to confirm the structure and are presented herein. The sequence and point of attachments of the sugars were determined by key 1,3-HMBC correlations between anomeric protons and their associated glycosylated carbons along with NOESY correlations (Fig. 2). The point of attachment of the penta-saccharide moiety with the aglycone was determined from the 1,3 HMBC correlation between the anomeric proton δΗ⁄C 4.22 (d, J = 7.3 Hz)/100.7 with C-3 at δC 76.2 of the aglycone. H3 of the aglycone, in turn, exhibited HMBC and NOESY correlations 170
Phytochemistry Letters 22 (2017) 167–173
R.R. Mosad et al.
Fig. 2. Key HMBC and COSY correlations for parquispiroside (1) and parquifuroside (2).
correlation of H-16 and C-15 confirmed the hydroxylation at C-15. The strong NOESY correlations between H-15, H-16 and H-17 (δΗ 2.17, dd, J = 11.0, 9.0 Hz) along with H-14 (δΗ 0.97, m) suggested the β-configuration of the hydroxy group introduced at C-15. The characteristic oxygenated methylene H-26 δH/C 3.78, 3.41 (m)/76.0 showed an HMBC correlations with the anomeric carbon at δC/H 104.6/4.30 (d, J = 7.8 Hz) and with the H-24 methine δH/C 1.78 (m)/35.0. These correlations confirmed the glycosylation at C-26 in which the presence of the common terminal glucose unit with β-configuration at the anomeric carbon (J = 7.8 Hz) was confirmed through the careful analysis of 13C NMR, HSQC, HMBC, 1H-1H NMR and NOESY correlations (Figs. 2 and 3). The absolute configuration of C-25 was assigned based on the analysis of the difference in chemical shift between H-26α and H-26β or Δab (Challinor et al., 2012). It was reported that the absolute configuration at C-25 is assigned as R configuration when Δab value is between 0.33-0.35 and assigned as S if Δab is in the range of 0.45-0.48 in deuterated methanol (Challinor et al., 2012). Compound 2 exhibited an Δab value of 0.37 ppm which is consistent with an assignment of R
Fig. 3. Key NOESY correlations of parquispiroside (1) and parquifuroside (2).
complete structure of 1 as 25(R)-3β [(O-β-D-glucopyranosyl-(1 → 3)-βD-glucopyranosyl-(1 → 2)-O-[β-D-xylopyranosyl-(1 → 3)-O-β-D-glucopyranosyl-(1 → 4)-β-D-galactopyranosyl)oxy]-5α, 15β, 22R, 25R-spirostan-3,15-diol (Fig. 1). Compound 2, parquifuroside, was obtained as a white amorphous solid. Positive ESIMS spectral data revealed a major ion at m/z 1432 (M +K)+ and the 13C NMR (MeOH, d4) spectrum contained 62 carbon resonances suggesting a molecular formula of C62H104O34. A furostanol with six sugars was suggested for 2 from the presence of the hemiacetal carbon resonance at δC 115.2 and six anomeric carbons at δC 105.0, 104.8, 104.5, 103.6, 102.6. The 1H NMR showed the four characteristic methyl groups resonances for the aglycone core at δΗ 1.05 (d, J = 6.6 Hz), 1.03 (s), 0.97 (d, J = 6.6 Hz) and 0.92 (s) in addition to three oxygenated methines at δΗ 3.69 (m), 4.12 (dd, J = 4.9, 3.7 Hz) and δΗ 4.43 (dd, J = 5.1, 3.9 Hz) which are correlated to the carbon resonances at δC 79.3, 79.8, and δC 84.0 respectively in the HSQC spectra along with an oxygenated methylene δH/C 3.78, 3.41 (m)/76.0. Detailed analysis of HMBC, 1H-1H-COSY and NOESY spectra enabled elucidation of the core furostanol moiety. The downfield-shifted carbon resonance at δC 84.0 was assigned to C-16 and suggested a hydroxylation at C-15, which was also downfield-shifted at δC 79.8. The 1 H-1H-COSY correlations between H-16 at δΗ 4.43 (dd, J = 5.1, 3.9 Hz) and H-15 at δΗ 4.12 (dd, J = 4.9, 3.7 Hz) as well as the HMBC
Table 2 Cytotoxic effect of the isolated compounds (IC50). IC50 [μM]
Parquispiroside (1) Parquifuroside (2) Capsicoside D (3) 22-OMe capsicoside (4) Benzyl primeveroside (5) Cisplatin
HeLa
HepG2
MCF-7
U87
7.7 ± 1.5 NA NA NT NA
7.2 ± 1.4 NA NA NT NA
14.1 ± 4.5 NA NA NT NA
3.3 ± 0.63 NA NA NT NA
39.2 ± 8.2
14.6 ± 5.9
7.3 ± 1.3
23.0 ± 5.6
NT: not tested; NA: not active, no cytotoxicity was observed at the tested concentrations. Cisplatin was used as a positive control with the following concentrations [HeLa (40 μM), HepG2 (20 μM), U-87 MG (25 μM), and MCF7 (10 μM)].
171
Phytochemistry Letters 22 (2017) 167–173
R.R. Mosad et al.
as the absolute configuration for C-25. The oxygenated methine at δC 79.3 was assigned to C-3 which is always β-oriented. The trans A/B junction configuration in 2 was assigned based on the NOESY correlation between H-3 (δΗ 3.69, m) and H-5 (δΗ 1.15, m). Based on these data and after careful analysis of all the HSQC and HMBC correlations, the agylcone is identified as 25R-26-[(β-D-glucopyranosyl)oxy]-(3β, 5α, 15β, 22R, 25R)-furostane-3,15,22-triol. The 1H NMR (methanol, d4) of 2 revealed the presence of six anomeric resonances at δΗ 4.40 (d, J = 7.8 Hz), 4.62 (d, J = 7.8 Hz), 4.65 (d, J = 7.8 Hz), 5.01 (d, J = 7.8 Hz), and δH 4.60 (d, J = 7.8 Hz) which are correlated to δC 102.6, 104.5, 104.8, 103.6 and δC 105.0 respectively in the 13C NMR spectra of 2. The anomeric resonance at δΗ 4.40 was assigned already to the terminal glucose unit attached to C-26 and hence, a penta-saccharide fragment is attached to C-3. Attachment of the pentasaccharide fragment to C-3 of the aglycone was confirmed through the strong 1,3-HMBC correlations between H-3 (δΗ 3.69, m) and H′ of the galactose unit at δΗ 4.40 (d, J = 7.8 Hz). Detailed analysis of the HMBC, 1H-1H-COSY, and NOESY correlations of the anomeric carbons and protons as well as literature NMR data (published in deuterated methanol) of similar polysaccharide fragments enabled us to confirm the sequence and structure of the pentasaccharide as shown in Fig. 1. The sugar part in 2 is identical to the sugar part in 1 and has been reported previously in several saponins isolated from several plant species with a confirmed D-configuration of all sugar moieties, and thus, we assumed the D-configuration for our sugars as well (Mimaki et al., 2002; Yahara et al., 1994; Maisashvili et al., 2012; Fattorusso et al., 2000). Based on that, the structure of the new compound 2 is 25R-26-[(β-D-glucopyranosyl)oxy]-(3β [(O-β-D-glucopyranosyl-(1 → 3)-β-D-glucopyranosyl-(1 → 2)-O-[β-D-xylopyranosyl-(1 → 3)-O-β-Dglucopyranosyl-(1 → 4)-β-D-galactopyranosyl)oxy], 5α, 15β, 22R, 25R)-furostane-3,15,22-triol (Fig. 1). Careful analysis of the 1H NMR (methanol, d4) of compound 3 suggest high similarity with the structure of 2 including identical hexasaccharide and furostanol steroidal fragments. Overlaying the 1H NMR spectrum of 2 and 3 (methanol, d4) revealed the absence of the H-15 resonances at δΗ 4.12 (dd, J = 4.9, 3.7 Hz) in the 1H NMR spectra of 3 which suggest that the hydroxylation at C-15 is missing in compound 3. Compound 3 was reported previously from seeds of capsicum and named capsicoside D (Iorizzi et al., 2002). The 1H NMR spectrum of compound 4 was identical to the 1H NMR spectra of 3 except an additional resonance for an OMe group at δΗ 3.31 was present. This compound was reported previously and named 22-OMe capsicoside D (Iorizzi et al., 2002). Compound 5 was obtained as a white amorphous solid. Positive ESIMS spectral data revealed a major ion at m/z 423 (M+Na)+ and the 13 C NMR (MeOH, d4) spectrum contained 18 carbon resonances, suggesting a molecular formula of C18H26O10. The 1H NMR showed the presence of an aromatic ring with one substitution [δΗ (7,45, m, 2H), (7.35, m, 2H) and (7.28, m, 1H)] in addition to two anomeric proton resonances at δΗ 4.36 (d, J = 7.9 Hz) and 4.37 (d, J = 7.8 Hz), which were correlated to the anomeric carbon resonances at δC 103.8 and δC 102.0 respectively in the HSQC spectrum of 5. The HMBC correlations of the methylene protons at δΗ 4.54 (d, J = 11.74 Hz), δH/C 4.69 (d, J = 11.98 Hz)/70.5 together with the aromatic ring carbon resonances (δC 138.1 and δC 128.4) and the anomeric carbon at δC 102⋅1 suggest that compound 5 is a benzyl glycoside of a disaccharide. The large J values (∼7 Hz) of the two anomeric proton resonances suggested a βconfiguration for both sugars. Careful analysis of the HMBC and 1H-1HCOSY of the two anomeric protons revealed that a benzylic group was bound to the C-1 position of a glucose unit, which additionally glycosylated at its C-6 position with a xylose unit. This compound is known as benzyl primeveroside. This is the first report of this compound from Cestrum parqui. Cytotoxicity evaluation of the isolated compounds was carried out against four human cell lines: HeLa, HepG2, U87, and MCF7 (Table 2). Only compound 1, parquispiroside, was moderately active, with IC50
values of 7.7, 7.2, 14.1, and 3.3 μM against HeLa, HepG2, U87, and MCF7 respectively. Author contributions RRM and AEW designed the isolation and the purification schemes and recorded the NMR data. AEW elucidated the structure of all isolated compounds and wrote the manuscript. MHA and ME designed and ran the biological evaluation. MTI and HMS are the master committee members of RRM thesis. MTI suggested working on Cestrum parqui. Funding sources The isolation and structure elucidation part was a self-funded project by RRM and AEW. Zewail City of Science and Technology startup fund for ME. Acknowledgments We would like to thank Zewail City for Science and Technology, Egypt, for supporting the biological evaluation of our compounds. All NMR analyses were completed at Kafr ElSheikh University, Egypt and we would like to thank them and express our deep appreciation to the NMR unit team: Professor Ahmed Khodeir, Dean of the Faculty of Science and the Unit Chair, Professor Adel Atya, former Chemistry Department Chair, Mrs. Rawda Emad and Mrs. Mayada, the machine technicians for their kind support and understanding. We also want to thank Dr. Ibrahim El Sherbiny, Professor of Material Sciences and Mr. Amr Hefnawy, Research Assistant, Zewail City of Science and Technology for their kind help with drying down our samples using the freeze dryer. Thanks to Michael W. Mullowney for assistance in copyediting of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.phytol.2017.09.022. References Agrawal, P.K., Jain, D.C., Pathak, A.K., 1995. NMR spectroscopy of steroidal sapogenins and steroidal saponins: an update. Magn. Reson. Chem. 33, 923–953. Agrawal, P.K., 2003. 25R/25S stereochemistry of spirostane-type steroidal sapogenins and steroidal saponins via chemical shift of geminal protons of ring-F. Magn. Reson. Chem. 41, 965–968. Baqui, F.T., Ali, A., Ahmad, V.Q., 2001. Two new spirostanol glycosides from Cestrum parqui. Helv. Chim. Acta 84, 3350–3356. Challinor, V.L., Piacente, S., De Voss, J.J., 2012. NMR assignment of the absolute configuration of C-25 in furostanol steroidal saponins. Steroids 77, 602–608. D'Abrosca, B., DellaGreca, M., Fiorentino, A., Monaco, P., Oriano, P., Temussi, F., 2004. Structure elucidation and phytotoxicity of C13 nor-isoprenoids from Cestrum parqui. Phytochemistry 65, 497–505. Fattorusso, E., Lanzotti, V., Taglialatela-Scafati, O., Di Rosa, M., Ianaro, A., 2000. Cytotoxic saponins from bulbs of Allium porrum L. J. Agric. Food Chem. 48, 3455–3462. Hayes, P.Y., Jahidin, A.H., Lehmann, R., Penman, K., Kitching, W., De Voss, J.J., 2008. Steroidal saponins from the roots of Asparagus racemosus. Phytochemistry 69, 796–804. Hu, K.F., Berenjian, S., Larsson, R., Gullbo, J., Nygren, P., Lovgren, T., Morein, B., 2010. Nanoparticulate Quillaja saponin induces apoptosis in human leukemia cell lines with a high therapeutic index. Int. J. Nanomed. 5, 51–62. Iorizzi, M., Lanzotti, V., Ranalli, G., De Marino, S., Zollo, F., 2002. Antimicrobial furostanol saponins from the seeds of capsicum annuum L. Var. acuminatum. J. Agric. Food Chem. 50, 4310–4316. Judd, W.S., Campbell, C.S., Kellogg, E.A., Stevens, P.F., 1999. Plant Systematics: A Phylogenetic Approach. Sinauer Associates, Sunderland. Lorent, J.H., Quetin-Leclercqb, J., Mingeot-Leclercq, M., 2014. The amphiphilic nature of saponins and their effects on artificial and biological membranes and potential consequences for red blood and cancer cells. Org. Biomol. Chem. 12, 8803–8822. Maisashvili, M.R., Kuchukhidze, D.K., Kikoladze, V.S., Gvazava, L.N., 2012. Steroidal glycosides of gitogenin from Allium rotundum. Chem. Nat. Comp. 48, 86–90. McLennan, M.W., Kelly, W.R., 1984. Cestrum Parqui (Green Cestrum) Poisoning in Cattle. Aust. Vet. J. 61, 289–291.
172
Phytochemistry Letters 22 (2017) 167–173
R.R. Mosad et al.
Sci. 51, 289. Sparg, S.G., Light, M.E., Staden, J.V., 2004. Biological activities and distribution of plant saponins. J. Ethnopharmcol. 94, 219–243. Tschesche, R., Wulff, G., 1963. Uper saponine der spirostanolreihe-IX, die constitution des digitonins. Tetrahedron Lett. 19, 621–634. Yahara, S., Ura, S., Sakamoto, C., Nohara, T., 1994. Steroidal glycosides frorm capsicum annuum. Phytochemistry 37, 831–835.
Mimaki, Y., Yokosuka, A., Sakuma, C., Sakagami, H., Sashid, Y., 2002. Spirostanol pentaglycosides from the underground parts of Polianthes tuberosa. J. Nat. Prod. 65, 1424–1428. Rejinold, N.S., Muthunarayanan, M., Muthuchelianb, K., Chennazhia, K.P., Naira, S.V., Jayakumar, R., 2011. Saponin-loaded chitosan nanoparticles and their cytotoxicity to cancer cell lines in vitro. Carbohydr. Polym. 84, 407–416. Silva, M., Mancinelli, P., Cheul, M., 1962. Chemical study of Cestrum parqui. J. Pharm.
173
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
New cytotoxic steroidal saponins from Cestrum parqui a
b
a
MARK a
b
Radwa R. Mosad , Mohamed H. Ali , Magda T. Ibrahim , Hala M. Shaaban , Marwan Emara , ⁎ Amir E. Wahbac, a b c
Department of Pharmacognosy, Faculty of Pharmacy For Girls, Al-Azhar University, Egypt Center of Aging and Associated Diseases, Zewail City of Science and Technology, Egypt Chemistry Department, Faculty of Science, Damietta University, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords: Cestrum parqui Steroidal saponins Spirostanol Furostanol Cytotoxicity Digalogenin
Two new steroidal saponins, 25(R)-3β [(O-β-D-glucopyranosyl-(1 → 3)-β-D-glucopyranosyl-(1 → 2)-O-[β-D-xylopyranosyl-(1 → 3)-O-β-D-glucopyranosyl-(1 → 4)-β-D-galactopyranosyl)oxy]-5α, 15β, 22R, 25R-spirostan3,15-diol (1, named parquispiroside) and 25R-26-[(β-D-glucopyranosyl)Oxy]-(3β [(O-β-D-glucopyranosyl(1 → 3)-β-D-glucopyranosyl-(1 → 2)-O-[β-D-xylopyranosyl-(1 → 3)-O-β-D-glucopyranosyl-(1 → 4)-β-D-galactopyranosyl)oxy], 5α, 15β, 22R, 25R)-furostane-3,15,22-triol (2, named parquifuroside), along with the known saponins, capsicoside D (3) and 22-OMe-capsicoside D (4) and the known glycoside, benzyl primeveroside (5), were isolated from the leaves of Cestrum parqui. The structures of these compounds were elucidated by careful analysis of 1D and 2D NMR spectra and ESIMS data. Parquispiroside (1) exhibited moderate inhibition of Hela, HepG2, U87, and MCF7 cell lines with IC50 values in the range of 3.3–14.1 μM.
1. Introduction Saponins are structurally complex glycosides composed of a nonpolar steroid, or triterpene, modified with one or more highly polar sugar moieties. The resulting amphiphilic physical properties of saponins provide for unique interactions with living cells. Several biological activities have been reported for saponins including: anticancer, antiviral, hypoglycemic, anti-inflammatory, heamolytic, and antifungal (Sparg et al., 2004). Specifically, saponins have the ability to bind the phospholipid bilayer and cholesterol to form pores in the cell membrane and cause cell lysis (Lorent et al., 2014). Several saponins have been shown to induce cell death by multiple mechanisms including apoptosis, autophagy, and necrosis (Lorent et al., 2014), suggesting a potential to evade drug-resistance. However, cancer treatment with saponins suffers the limitation of a narrow therapeutic window due to potential toxicity. The emerging field of nanoformulation could help to increase the therapeutic window and hence open the door for the possibility of using pure and/or mixtures of saponins in clinical applications (Rejinold et al., 2011; Hu et al., 2010). As a part of our program to identify anticancer drug leads from the Egyptian environment, we investigated the methanolic extract of the leaves of Cestrum parqui, a shrub commonly known as the green cestrum. While many plants of the genus Cestrum, the second largest genus of the Solanaceae family (Judd et al., 1999), have been explored extensively, natural products from Cestrum parqui occur in little reports to ⁎
date. The plant is commonly known for its toxicity to cattle (McLennan and Kelly, 1984). Saponins are the major group of natural products isolated from Cestrum parqui (Baqui et al., 2001; D'Abrosca et al., 2004; Silva et al., 1962). In this study, we report the isolation, structure elucidation and anticancer evaluation of two new steroidal saponins along with three known compounds from the leaves of Cestrum parqui. 2. Material and methods 2.1. General experimental procedure Nuclear Magnetic Resonance spectra (1D and 2D) were recorded with a Bruker Ascend™ spectrometer (Bruker Daltonics, Bremen, Germany) at 400 MHz for proton, and 100 MHz for carbon. The data obtained were processed using ACD-NMR processor software ver. 12.01. The solvents used were CD3OD and DMSO d6. Chemical shift data was reported in parts per million (ppm) on the δ scale; coupling constants (J) were expressed in Hz. LC–MS/MS was carried out using a Waters TQ detector, Acquity ultra performance LC binary solvent manager and auto sampler operated by Mass Lynx software. Pre-coated silica gel plates (TLC) F254 (20 × 20 cm) were used for analytical separation, using ascending technique and suitable solvent systems with visualization using p-anisaldehyde/H2SO4 and vanillin solution. Normal phase chromatography was carried out using silica gel G60-230 (Merck, Darmstadt, Germany) packed by the wet method in the stated solvent.
Corresponding author. E-mail address: [email protected] (A.E. Wahba).
http://dx.doi.org/10.1016/j.phytol.2017.09.022 Received 20 June 2017; Received in revised form 24 September 2017; Accepted 27 September 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 22 (2017) 167–173
R.R. Mosad et al.
Sephadex LH-20 column chromatography was carried out using sephadex (SIGMA-ALDRICH, Missouri, USA). Reversed phase chromatography was performed using phase-bonded octadecylsilyl-silica gel (RPC18, BAKERBOND® Octadecyl, C18) 40 μm, Prep LC packing (Phillipsburg, NJ, USA).
Table 1 NMR spectroscopic data for parquispiroside(1) and parquifuroside (2). Position
Parquispiroside (1) (DMSO, d6) δC,
2.2. Plant materials Fresh leaves of Cestrum parqui L’Hér were collected in November 2014 from plants cultivated in El-Kanater El-Khairia gardens and Ahmed Alaa’s garden in Shebeen Elkom, Menoufia, Egypt. The plant was identified and confirmed by Prof. Abd El-Hamid Khedr, Department of Botany, Faculty of Science, Damietta University, Damietta, Egypt. A voucher specimen was kept at the Department of Pharmacognosy, Faculty of Pharmacy for Girls, AlAzhar University and was given the reference number (CP01). 2.3. Extraction and isolation The air dried, powdered leaves (1 kg) were extracted by maceration with cold distilled CH3OH (10 L) for four days. The methanolic extract was concentrated under reduced pressure to constant weight (≈150 g). The dried methanolic residue was dissolved in the least volume of MeOH, loaded onto 100 g silica gel and chromatographed over a silica gel column (150 × 5 cm, i.d., 500 g) elution started using 100% petroleum ether, then increasing polarity gradually using petroleum ether: methylene chloride, (wa EtOAc)1 till 100% CH3OH (10% increment) and then the column was washed with n-butanol. Fractions of 100 mL each were collected, concentrated, and monitored by TLC with an appropriate solvent system. Five pools were collected. Pool 4 (fractions eluted with 100% wa EtOAc up to 100% MeOH were combined, 22.1 g) was chromatographed over silica gel column (100 × 2.5 cm i.d., 160 g) and eluted with pet. ether: wa EtOAc mixtures with gradual increase of polarity (10% increments) till 100% wa EtOAc then wa EtOAc: CH3OH up to 100% CH3OH. 800 fractions were collected (20 mL each) and monitored by TLC. Fractions 701–738 were combined and compound 1 was precipitated out as a pure white amorphous solid. Fractions 750–800 were combined and further purified over RP-C18 column with an isocratic solvent system (MeOH:H2O 1:9, 10 mL fraction size). Fractions 5–20 were combined and purified over a preparative TLC plate with a mixture of 12:8:8:2 (wa EtOAc: DCM: MeOH: H2O) as the eluting system to give compound 5. Pool 5 (n-butanol fraction, 9 g) was fractionated over silica gel with wa EtOAc to MeOH step gradient to produce four fractions. Fraction 4 (100% MeOH, 1.2 g) was further purified over a RP-C18 column using the isocratic solvent system, MeOH:H2O 3:7 that gave pure compounds 2–4.
type
1
36.2, CH2
2
29.6, CH2
3 4
76.2, CH 33.8, CH2
5 6
44.0, CH 28.1, CH2
7
30.3, CH2
8 9 10 11
30.6, 53.7, 35.2, 20.6,
CH CH C CH2
δH (J Hz)
δC,type
δH (J Hz)
1⋅62 0.92 1⋅74 1.38 3.54 1.61 1.17 1.01 1.60 1.24 1.76 1.41 1.76 0.68 – 1.43 1.28
38.0, CH2
1.75 (m) 1.06 (m) 1.7 (m) 1.4 (m) 3.69 (m) 1.57 (m) 1.33 (m) 1.15 (m) 1.92 (m) 1.59 (m) 2.00 (m) 0.99 (m) 1.8 (m) 0.86 (m) –
(m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m)
12
2.3.1. Parquispiroside (1); 25(R)-3β [(O-β-D-glucopyranosyl-(1 → 3)-β-Dglucopyranosyl-(1 → 2)-O-[β-D-xylopyranosyl-(1 → 3)-O-β-Dglucopyranosyl-(1 → 4)-β-D-galactopyranosyl)oxy]-5α, 15β, 22R, 25Rspirostan-3,15-diol Amorphous white solid (MeOH), 5.7 mg; 1H NMR (DMSO, d6, 400 MHz) and 13C NMR (DMSO, d6, 400 MHz) see Table 1; ESI–MS (+ve) m/z 1242 [M+K]+ 2.3.2. Parquifuroside (2): 25R-26-[(β-D-glucopyranosyl)Oxy]-(3β [(O-βD-glucopyranosyl-(1 → 3)-β-D-glucopyranosyl-(1 → 2)-O-[β-Dxylopyranosyl-(1 → 3)-O-β-D-glucopyranosyl-(1 → 4)-β-Dgalactopyranosyl)oxy], 5α, 15β, 22R, 25R)-furostane-3,15,22-triol Amorphous white solid (MeOH), 7.0 mg; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 400 MHz) see Table 1; ESI–MS (+ve) m/z 1432 [M+K]+ 1 Commercial ethyl acetate was mixed with 3% (v/v) water and 1% (v/v) acetic acid and shake well until clear solution was observed.
Parquifuroside (2) (CD3OD)
29.7, CH2 79.3, CH 28.5, CH2 44.8, CH 29.0, CH2 31.5, CH2 35.0, 54.9, 35.9, 23.8,
CH CH C CH2
36.3, CH2
13 14 15 16 17 18 19 20 21 22 23
40.4, C 58.9, CH 68.6, CH 82.3, CH 60.6, CH 18.2, CH3 11.8, CH3 41.7, CH 14.0, CH3 109.1, C 31.1, CH2
– 0.93 3.93 4.13 1.80 0.95 0.80 1.83 0.88 – 1.86
24
28.8, CH2
25 26
29.5, CH 65.6, CH2
1.77 1.40 1.53 3.43 3.10 0.75
27
16.9, CH3
1 2 3 4 5 6
Gal-I 100.7, CH 71.2, CH 73.3, CH 78.4, CH 76.2, CH 59.3, CH2
1 2 3 4 5 6
Glu-I 102.8, CH 78.3, CH 84.6, CH 68.5, CH 76.2, CH 60.5, CH2
1 2 3 4 5 6
Glu-II 101.1, CH 72.7, CH 86.9, CH 67.9, CH 76.2, CH 60.8, CH2
1 2 3 4 5 6
Glu-III 103.6, CH 73.6, CH 75.8, CH 69.9, CH 76.6, CH 61.0, CH2
4.22 3.24 3.35 3.78 3.01 3.72 3.40
(m) (dd, 4.9, 3.7) (dd, 5.38, 5.38) (m) (s) (s) (m) (d, 6.0) (m), 1.61 (m) (m) (m) (m) (m) (m) (d, 6.0) (d, 7.6) (m) (m) (m) (m) (m) (m)
4.45 (d, 7.6) 3.61 (m) 3.62 (m) 3.13 (m) 3.12 (m) 3.7 (m) 3.38 (m) 4.87 (d, 7.6) 3.23 (m) 3.37 (m) 3.26 (m) 3.10 (m) 3.4 (m) 3.68 (m) 4.32 3.06 3.18 3.10 3.27 3.67 3.34
(d, 7.8) (m) (m) (m) (m) (m) (m)
44.7, C 59.5, CH 79.8, CH 84.0, CH 61.3, CH 41.8, CH3 14.2, CH3 13.1, CH 12.5, CH3 115.2, C 35.2, CH2 30.3, CH2 35.0, CH 76.0, CH2 17.2, CH3 Gal-I 102.6, CH 73.0, CH 75.2, CH 79.8, CH 75.5, CH 61.0, CH2 Glu-I 104.5, CH 80.7, CH 87.5, CH 70.4, CH 77.3, CH 61.0, CH2 Glu-II 103.6, CH 75.2, CH 87.4, CH 70.0, CH 78.0, CH 62.5, CH2 Glu-III 105.0, CH 75.5, CH 77.8, CH 71.5, CH 77.0, CH 60.0, CH2
1.8 (m) 1.72 (m) – 0.97 (m) 4.12 (dd, 4.9, 3.6) 4.43 (dd, 5.1, 3.9) 2.17 (dd, 11.3, 9) 2.10 (m) 1.05 (d, 6.6) 1.03 (s) 0.92 (s) – 1.85 (m) 1.74 (m) 1.71 (m) 1.1 (m) 1.78 (m) 3.78 (m) 3.41 (m) 0.97 (d, 6.4) 4.40 3.64 3.54 4.02 3.51 3.90 3.68
(d, 7.7) (m) (m) (m) (m) (m) (m)
4.62 3.80 3.74 3.32 3.39 3.90 3.68
(d, 7.6) (m) (m) (m) (m) (m) (m)
5.0 (d, 7.7) 3.43 (m) 3.60 (m) 3.48 (m) 3.74 (m) 3.83 (m) 3.65 (m) 4.60 3.32 3.37 3.66 3.34 3.87 3.69
(d, 7.7) (m) (m) (m) (m) (m) (m)
(continued on next page)
168
Phytochemistry Letters 22 (2017) 167–173
R.R. Mosad et al.
prepared in DMSO (Sigma Aldrich, USA). Working concentrations contained less than 0.15% (v/v) DMSO and the same amount of DMSO was added to the control group as the experimental group. Cisplatin positive control was used at the different concentrations known to induce cytotoxic effect in different cell lines [HeLa (40 μM), HepG2 (20 μM), U-87 MG (25 μM), and MCF7 (10 μM)]. All treated and untreated cultures were incubated for 48 h followed by addition of CCK-8 solution (10 μL/well), incubation for two additional hours, and absorbance measurement at 450 nm using microplate reader (FLUOstar Omega, BMG LABTECH GmbH, Germany). The number of living cells is directly proportional to the amount of formazan dye generated by dehydrogenases in these cells. The percentage of viable cells was calculated according to the following formula:
Table 1 (continued) Position
Parquispiroside (1) (DMSO, d6) δC,
type
1 2 3 4 5
Xyl 103.0, CH 75.7, CH 76.2, CH 69.1, CH 65.9, CH2
1 2 3 4 5 6
– – – – – –
δH (J Hz)
4.51 3.10 3.22 3.34 3.44 3.24
(d, 7.6) (m) (m) (m) (m) (m)
– – – – – –
Parquifuroside (2) (CD3OD) δC,type Xyl 104.8, CH 74.0, CH 77.9, CH 69.3, CH 67.0, CH2
104.6, CH 75.0, CH 77.6, CH 71.3, CH 76.6, CH 61.2, CH2
δH (J Hz)
4.65 3.27 3.38 3.32 3.95 3.27
(d, 7.7) (m) (m) (m) (m) (m)
Glu-IV 4.30 (d, 7.7) 3.2 (m) 3.37 (m) 3.31 (m) 3.35 (m) 3.95 (m) 3.68 (m)
Cell Viability (%) =
A sample − Ablank A control − Ablank
× 100
2.6. Statistical analysis Data from three biological replicate experiments with three technical replicate each were expressed as mean ± SE. Statistical analysis was carried out using SigmaPlot 13 (Systat software, Inc, USA). The IC50 was calculated using Microcal Origin software program (Microcal software Inc., USA).
2.3.3. Capsicoside D (3) Amorphous white solid (MeOH), 3.0 mg; 1H NMR (CD3OD, 400 MHz): δ 5.0 (1H, d, J = 7.7), 4.65 (1H, d, J = 7.7), 4.62 (1H, d, J = 7.6), 4.60 (1H, d, J = 7.7), 4.40 (1H, d, J = 7.7), 4.30 (1H, d, J = 7.7), 3.78 (1H, m), 3.69 (1H, m), 3.41 (1H, m), 1.05 (3H, d, J = 6.6), 1.03 (3H, s),0.97 (3H, d, J = 6.4), 0.92 (3H, s).
3. Results and discussion
2.3.4. 22-OMe capsicoside D (4) Amorphous white solid (MeOH), 2.3 mg; 1H NMR (CD3OD, 400 MHz): δ 5.0 (1H, d, J = 7.7), 4.65 (1H, d, J = 7.7), 4.62 (1H, d, J = 7.6), 4.60 (1H, d, J = 7.7), 4.40 (1H, d, J = 7.7), 4.30 (1H, d, J = 7.7), 3.78 (1H, m), 3.69 (1H, m), 3.41 (1H, m), 3.23 (3H, s), 1.05 (3H, d, J = 6.6), 1.03 (3H, s),0.97 (3H, d, J = 6.4), 0.92 (3H, s).
Compound 1, parquispiroside, was obtained as a white solid. A molecular formula of C56H92O28 was proposed for 1 from the ESI–MS spectrum, which contained a major ion at m/z 1242 [M+K]+ and was supported by the presence of 56 carbon resonances in the 13C NMR spectrum. The 1H NMR revealed the four methyl resonances typical of the steroid class, two of which were secondary at δH 0.75 (d, J = 6.1 Hz) and δH 0.88 (d, J = 6.1 Hz) while the remaining two were quaternary at δΗ 0.81 and δΗ 0.95. Five anomeric protons were also observed at δΗ 4.22 (d, J = 7.3 Hz), 4.32 (d, J = 7.8 Hz), 4.45 (d, J = 7.0 Hz), 4.51 (d, J = 7.6 Hz), and 4.86 (d, J = 7.6 Hz). The 13C NMR and DEPT135 spectra of 1 confirmed the steroidal glycoside nature of the compound in which 29 out of the 56 resonances were assigned for the penta-saccharide fragment. The remaining carbon resonances were assigned to the aglycone, which included the characteristic acetal carbon resonance for a spirostanol at δC 109.1 (C-22) (Agrawal et al., 1995) and three oxygenated methines at δC 82.3, 76.2 and 68.6. The downfield shifts of the resonances for C-15 and the C-16 methine to δC 68.6 and δC 82.8, respectively, suggested a hydroxylation at C-15. The HMBC and 1H-1H–COSY correlations of the methine proton H-16 at δH 4.13 (d, J = 5.4, 5.3 Hz) with carbon resonance at δC 68.6 and H-15 at δΗ 3.93 (m) confirmed the hydroxylation at C-15. The relative configuration of the newly introduced hyroxy group at C-15 is assigned as β orientation based on the strong NOESY correlations between H-15 and Me-21 (δΗ 0.88, d, J = 6.1 Hz), H-16 (δΗ 4.13, dd, J = 5.4, 5.3 Hz), H-17 (δΗ 1.83, m) and H-14 (δΗ 0.93, m). Mutual NOESY correlations between protons attached to rings D and E (H-14, H-16 and H-17) of the steroidal core confirmed the D/E cis junction. The assignment of the F ring was based on the HMBC correlations between the Me-27 protons (δH/C 0.75 (d, J = 6.1 Hz)/16.9) and the oxygenated methylene C-26 (δC 65.6), the C-25 methine (δC 29.5) and C-24 (δC 28.8). The absolute configuration of C-25 is assigned as R based on the chemical shift of Me-27 at δΗ⁄C 0.75/16.9 along with the downfield chemical shifts of C-24 (δC 28.8), C-25 (δC 29.5) and C-26 (δC 65.6) (Agrawal, 2003). The common R-configuration at C-22 is assigned based on the comparison of the chemical shifts of the F ring carbon resonances with the published, yet more rare S-configuration (Hayes et al., 2008). The NOESY correlation between H-5 (δH 1.01 (m)) and H-3 (δH 3.54 (m)) (Fig. 3) along with the carbon chemical shifts of C-5 (δC 44.72), C-
2.3.5. Benzyl primeveroside (5) Amorphous white solid (MeOH), 8.2 mg; 1H NMR (CD3OD, 400 MHz): δ 7.50–7.25 (5H, m), 4.92 (1Η, d, J = 12.0), 4.66 (1H, d, J = 12.0), 4.36 (1H, d, J = 7.6), 4.34 (1H, d, J = 7.6), 4.10 (1H, dd, J = 11.2, 2.1), 3.85 (1H, dd, J = 12.5, 3.4), 3.80 (1H, dd, J = 11.5, 5.9), 3.68 (1H, dd, J = 6.2, 3.2); 13C NMR (CD3OD, 100 MHz): 139.3, 129.4, 129.3, 128.8, 105.4, 103.6, 78.1, 77.2, 75.3, 74.4, 72.6, 72.1, 71.9, 69.7, 69.6, 66.8; ESI–MS (+ve) m/z 425 [M+Na]+ 2.4. Cell lines and in vitro culture conditions Four human cell lines of different cancer origin [HeLa Cells (cervical adenocarcinoma, CCL-2, ATCC, USA), U-87 MG (Glioblastoma, TB-14, ATCC), Hep G2 (HB-8065, ATCC), and MCF7 (Breast adenocarcinoma, HTB-22, ATCC)] were used. HeLa and HepG2 were cultured in RPMI 1640 (12-702F, Lonza, Verviers, Belgium) media, while U-87 MG and MCF7 were cultured in DMEM/F12 (BE12-719F, Lonza) and DMEM (BE12-604F, Lonza) media, respectively. All cells were maintained as monolayer cultures supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, USA) and L-glutamine (BE12-604F, Lonza) in a humidified atmosphere of 5% CO2 in air at 37 °C. No antibiotics or antimycotics were added. 2.5. Cell proliferation assay and cytotoxicity assay The cell proliferation assay and cytotoxicity assays was carried out using Cell counting kit-8 (CCK-8, Code: CK04, Tokyo, Japan). Cells were cultured in a flat bottom 96-microwell plate (4 × 103 cells/100 μL culture media/well) and incubated overnight. Cell lines were treated with the purified compounds at different concentrations (1 μM, 5 μM, 10 μM, 25 μM, and 50 μM) and compared with untreated cell line negative controls. Stock concentrations of the pure compounds were 169
Phytochemistry Letters 22 (2017) 167–173
R.R. Mosad et al.
Fig. 1. Structures of the isolated compounds from Cestrum parqui.
with the anomeric proton at δΗ⁄C 4.22(d, J = 7.34 Hz)/100.7 of the galactose unit. The strong NOESY correlations between H-1′ (δΗ 4.22) and H-3′ (δΗ 3.35) and H-3′ (δΗ 3.35) and H-4′ (δΗ 3.78) confirmed that a galactose unit is attached to the C-3 of the aglycone with a glycosylation at C-4′ (δc 78.4). H-4′ at δH/C (3.78, m)/78.4 of Gal showed HMBC and NOESY correlations with the anomeric carbon at δC/H 102.8/4.45 (d, J = 7.0 Hz) which is assigned to an inner glucose with glycosylation at C-2′′ (δC/H 78.3/3.61 (m)) and C-3′′ (δC/H 84.6/3.62 (m)). The characteristic downfield oxygenated methylene carbon at δC 65.9 for a pentose unit, in addition to the 1H-1H-COSY, HMBC and NOESY correlations of the anomeric proton δΗ⁄C 4.51(d, J = 7.6 Hz)/ 103.0 confirmed a terminal xylose unit to be attached to C-3′′ of GluI. Another hexose unit is attached to C-2′′ of GluI based on the HMBC correlation between H-2′′ with the anomeric carbon δC/H 101.1/4.86 (d, J = 7.6 Hz). Interestingly, this unit was reported to be a galactose in digalonin (Tschesche and Wulff, 1963). However, careful analysis of NOESY correlations of H-3′′′′ (δH/C 3.37 (m)/86.9) revealed strong NOESY correlations with the anomeric proton H-1′′′′ at δΗ 4.86 (d, J = 7.6 Hz) and C2′′′′-OH (δΗ 5.35, d, J = 5.4 Hz) and missing the key NOESY correlation with H-4′′′′ at δΗ 3.25 (m). This suggests that the hydroxy group at C-4′′′′ is at the β-orientation and thus a glucose unit is present (GluII). A terminal glucose unit is attached to C-3′′′′ of GluII in which H-3′′′′ showed an 1,3-HMBC correlations with the anomeric C1′′′′′ of the terminal glucose at δC/H 103.6/4.32. Therefore, analysis of the data in combination with literature comparison confirmed the
7 (δC 29.5) and C-19 (δC 11.8), which they absorbed similarly to the published chemical shifts of trans A/B saponins, suggest the trans fusion between A and B rings (Agrawal et al., 1995). Based on these NMR analyses, the aglycone was identified as (3β, 5α, 15β, 22R, 25R)spirostan-3,15-diol. This aglycone has been reported previously in 1963 (Tschesche and Wulff, 1963), named digalogenin, without NMR data. Thus, we report the first complete NMR assignment of 3-glycosylated digalogenin in DMSO, d6. The 13C NMR of 1 revealed the presence of 5 anomeric carbons at δC 100.7 (Gal), 102.8 (GluI), 103.8 (Xyl), 101.1 (GluII), and 103.6 (GluIII) in which they were correlated to δΗ 4.22 (d, J = 7.34 Hz), 4.45 (d, J = 7.0 Hz), 4.51 (d, J = 7.6 Hz), 4.86 (d, J = 7.6 Hz) and 4.32 (d, J = 7.8 Hz) respectively in the 1H NMR spectra of 1. The large J values (∼7 Hz) of all the anomeric proton resonances suggested the β-configuration of the five anomeric carbons. Unfortunately, there is no NMR data reported for similar saponins dissolved in deuterated DMSO to use for comparison. Thus, all correlations were carefully analyzed in order to confirm the structure and are presented herein. The sequence and point of attachments of the sugars were determined by key 1,3-HMBC correlations between anomeric protons and their associated glycosylated carbons along with NOESY correlations (Fig. 2). The point of attachment of the penta-saccharide moiety with the aglycone was determined from the 1,3 HMBC correlation between the anomeric proton δΗ⁄C 4.22 (d, J = 7.3 Hz)/100.7 with C-3 at δC 76.2 of the aglycone. H3 of the aglycone, in turn, exhibited HMBC and NOESY correlations 170
Phytochemistry Letters 22 (2017) 167–173
R.R. Mosad et al.
Fig. 2. Key HMBC and COSY correlations for parquispiroside (1) and parquifuroside (2).
correlation of H-16 and C-15 confirmed the hydroxylation at C-15. The strong NOESY correlations between H-15, H-16 and H-17 (δΗ 2.17, dd, J = 11.0, 9.0 Hz) along with H-14 (δΗ 0.97, m) suggested the β-configuration of the hydroxy group introduced at C-15. The characteristic oxygenated methylene H-26 δH/C 3.78, 3.41 (m)/76.0 showed an HMBC correlations with the anomeric carbon at δC/H 104.6/4.30 (d, J = 7.8 Hz) and with the H-24 methine δH/C 1.78 (m)/35.0. These correlations confirmed the glycosylation at C-26 in which the presence of the common terminal glucose unit with β-configuration at the anomeric carbon (J = 7.8 Hz) was confirmed through the careful analysis of 13C NMR, HSQC, HMBC, 1H-1H NMR and NOESY correlations (Figs. 2 and 3). The absolute configuration of C-25 was assigned based on the analysis of the difference in chemical shift between H-26α and H-26β or Δab (Challinor et al., 2012). It was reported that the absolute configuration at C-25 is assigned as R configuration when Δab value is between 0.33-0.35 and assigned as S if Δab is in the range of 0.45-0.48 in deuterated methanol (Challinor et al., 2012). Compound 2 exhibited an Δab value of 0.37 ppm which is consistent with an assignment of R
Fig. 3. Key NOESY correlations of parquispiroside (1) and parquifuroside (2).
complete structure of 1 as 25(R)-3β [(O-β-D-glucopyranosyl-(1 → 3)-βD-glucopyranosyl-(1 → 2)-O-[β-D-xylopyranosyl-(1 → 3)-O-β-D-glucopyranosyl-(1 → 4)-β-D-galactopyranosyl)oxy]-5α, 15β, 22R, 25R-spirostan-3,15-diol (Fig. 1). Compound 2, parquifuroside, was obtained as a white amorphous solid. Positive ESIMS spectral data revealed a major ion at m/z 1432 (M +K)+ and the 13C NMR (MeOH, d4) spectrum contained 62 carbon resonances suggesting a molecular formula of C62H104O34. A furostanol with six sugars was suggested for 2 from the presence of the hemiacetal carbon resonance at δC 115.2 and six anomeric carbons at δC 105.0, 104.8, 104.5, 103.6, 102.6. The 1H NMR showed the four characteristic methyl groups resonances for the aglycone core at δΗ 1.05 (d, J = 6.6 Hz), 1.03 (s), 0.97 (d, J = 6.6 Hz) and 0.92 (s) in addition to three oxygenated methines at δΗ 3.69 (m), 4.12 (dd, J = 4.9, 3.7 Hz) and δΗ 4.43 (dd, J = 5.1, 3.9 Hz) which are correlated to the carbon resonances at δC 79.3, 79.8, and δC 84.0 respectively in the HSQC spectra along with an oxygenated methylene δH/C 3.78, 3.41 (m)/76.0. Detailed analysis of HMBC, 1H-1H-COSY and NOESY spectra enabled elucidation of the core furostanol moiety. The downfield-shifted carbon resonance at δC 84.0 was assigned to C-16 and suggested a hydroxylation at C-15, which was also downfield-shifted at δC 79.8. The 1 H-1H-COSY correlations between H-16 at δΗ 4.43 (dd, J = 5.1, 3.9 Hz) and H-15 at δΗ 4.12 (dd, J = 4.9, 3.7 Hz) as well as the HMBC
Table 2 Cytotoxic effect of the isolated compounds (IC50). IC50 [μM]
Parquispiroside (1) Parquifuroside (2) Capsicoside D (3) 22-OMe capsicoside (4) Benzyl primeveroside (5) Cisplatin
HeLa
HepG2
MCF-7
U87
7.7 ± 1.5 NA NA NT NA
7.2 ± 1.4 NA NA NT NA
14.1 ± 4.5 NA NA NT NA
3.3 ± 0.63 NA NA NT NA
39.2 ± 8.2
14.6 ± 5.9
7.3 ± 1.3
23.0 ± 5.6
NT: not tested; NA: not active, no cytotoxicity was observed at the tested concentrations. Cisplatin was used as a positive control with the following concentrations [HeLa (40 μM), HepG2 (20 μM), U-87 MG (25 μM), and MCF7 (10 μM)].
171
Phytochemistry Letters 22 (2017) 167–173
R.R. Mosad et al.
as the absolute configuration for C-25. The oxygenated methine at δC 79.3 was assigned to C-3 which is always β-oriented. The trans A/B junction configuration in 2 was assigned based on the NOESY correlation between H-3 (δΗ 3.69, m) and H-5 (δΗ 1.15, m). Based on these data and after careful analysis of all the HSQC and HMBC correlations, the agylcone is identified as 25R-26-[(β-D-glucopyranosyl)oxy]-(3β, 5α, 15β, 22R, 25R)-furostane-3,15,22-triol. The 1H NMR (methanol, d4) of 2 revealed the presence of six anomeric resonances at δΗ 4.40 (d, J = 7.8 Hz), 4.62 (d, J = 7.8 Hz), 4.65 (d, J = 7.8 Hz), 5.01 (d, J = 7.8 Hz), and δH 4.60 (d, J = 7.8 Hz) which are correlated to δC 102.6, 104.5, 104.8, 103.6 and δC 105.0 respectively in the 13C NMR spectra of 2. The anomeric resonance at δΗ 4.40 was assigned already to the terminal glucose unit attached to C-26 and hence, a penta-saccharide fragment is attached to C-3. Attachment of the pentasaccharide fragment to C-3 of the aglycone was confirmed through the strong 1,3-HMBC correlations between H-3 (δΗ 3.69, m) and H′ of the galactose unit at δΗ 4.40 (d, J = 7.8 Hz). Detailed analysis of the HMBC, 1H-1H-COSY, and NOESY correlations of the anomeric carbons and protons as well as literature NMR data (published in deuterated methanol) of similar polysaccharide fragments enabled us to confirm the sequence and structure of the pentasaccharide as shown in Fig. 1. The sugar part in 2 is identical to the sugar part in 1 and has been reported previously in several saponins isolated from several plant species with a confirmed D-configuration of all sugar moieties, and thus, we assumed the D-configuration for our sugars as well (Mimaki et al., 2002; Yahara et al., 1994; Maisashvili et al., 2012; Fattorusso et al., 2000). Based on that, the structure of the new compound 2 is 25R-26-[(β-D-glucopyranosyl)oxy]-(3β [(O-β-D-glucopyranosyl-(1 → 3)-β-D-glucopyranosyl-(1 → 2)-O-[β-D-xylopyranosyl-(1 → 3)-O-β-Dglucopyranosyl-(1 → 4)-β-D-galactopyranosyl)oxy], 5α, 15β, 22R, 25R)-furostane-3,15,22-triol (Fig. 1). Careful analysis of the 1H NMR (methanol, d4) of compound 3 suggest high similarity with the structure of 2 including identical hexasaccharide and furostanol steroidal fragments. Overlaying the 1H NMR spectrum of 2 and 3 (methanol, d4) revealed the absence of the H-15 resonances at δΗ 4.12 (dd, J = 4.9, 3.7 Hz) in the 1H NMR spectra of 3 which suggest that the hydroxylation at C-15 is missing in compound 3. Compound 3 was reported previously from seeds of capsicum and named capsicoside D (Iorizzi et al., 2002). The 1H NMR spectrum of compound 4 was identical to the 1H NMR spectra of 3 except an additional resonance for an OMe group at δΗ 3.31 was present. This compound was reported previously and named 22-OMe capsicoside D (Iorizzi et al., 2002). Compound 5 was obtained as a white amorphous solid. Positive ESIMS spectral data revealed a major ion at m/z 423 (M+Na)+ and the 13 C NMR (MeOH, d4) spectrum contained 18 carbon resonances, suggesting a molecular formula of C18H26O10. The 1H NMR showed the presence of an aromatic ring with one substitution [δΗ (7,45, m, 2H), (7.35, m, 2H) and (7.28, m, 1H)] in addition to two anomeric proton resonances at δΗ 4.36 (d, J = 7.9 Hz) and 4.37 (d, J = 7.8 Hz), which were correlated to the anomeric carbon resonances at δC 103.8 and δC 102.0 respectively in the HSQC spectrum of 5. The HMBC correlations of the methylene protons at δΗ 4.54 (d, J = 11.74 Hz), δH/C 4.69 (d, J = 11.98 Hz)/70.5 together with the aromatic ring carbon resonances (δC 138.1 and δC 128.4) and the anomeric carbon at δC 102⋅1 suggest that compound 5 is a benzyl glycoside of a disaccharide. The large J values (∼7 Hz) of the two anomeric proton resonances suggested a βconfiguration for both sugars. Careful analysis of the HMBC and 1H-1HCOSY of the two anomeric protons revealed that a benzylic group was bound to the C-1 position of a glucose unit, which additionally glycosylated at its C-6 position with a xylose unit. This compound is known as benzyl primeveroside. This is the first report of this compound from Cestrum parqui. Cytotoxicity evaluation of the isolated compounds was carried out against four human cell lines: HeLa, HepG2, U87, and MCF7 (Table 2). Only compound 1, parquispiroside, was moderately active, with IC50
values of 7.7, 7.2, 14.1, and 3.3 μM against HeLa, HepG2, U87, and MCF7 respectively. Author contributions RRM and AEW designed the isolation and the purification schemes and recorded the NMR data. AEW elucidated the structure of all isolated compounds and wrote the manuscript. MHA and ME designed and ran the biological evaluation. MTI and HMS are the master committee members of RRM thesis. MTI suggested working on Cestrum parqui. Funding sources The isolation and structure elucidation part was a self-funded project by RRM and AEW. Zewail City of Science and Technology startup fund for ME. Acknowledgments We would like to thank Zewail City for Science and Technology, Egypt, for supporting the biological evaluation of our compounds. All NMR analyses were completed at Kafr ElSheikh University, Egypt and we would like to thank them and express our deep appreciation to the NMR unit team: Professor Ahmed Khodeir, Dean of the Faculty of Science and the Unit Chair, Professor Adel Atya, former Chemistry Department Chair, Mrs. Rawda Emad and Mrs. Mayada, the machine technicians for their kind support and understanding. We also want to thank Dr. Ibrahim El Sherbiny, Professor of Material Sciences and Mr. Amr Hefnawy, Research Assistant, Zewail City of Science and Technology for their kind help with drying down our samples using the freeze dryer. Thanks to Michael W. Mullowney for assistance in copyediting of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.phytol.2017.09.022. References Agrawal, P.K., Jain, D.C., Pathak, A.K., 1995. NMR spectroscopy of steroidal sapogenins and steroidal saponins: an update. Magn. Reson. Chem. 33, 923–953. Agrawal, P.K., 2003. 25R/25S stereochemistry of spirostane-type steroidal sapogenins and steroidal saponins via chemical shift of geminal protons of ring-F. Magn. Reson. Chem. 41, 965–968. Baqui, F.T., Ali, A., Ahmad, V.Q., 2001. Two new spirostanol glycosides from Cestrum parqui. Helv. Chim. Acta 84, 3350–3356. Challinor, V.L., Piacente, S., De Voss, J.J., 2012. NMR assignment of the absolute configuration of C-25 in furostanol steroidal saponins. Steroids 77, 602–608. D'Abrosca, B., DellaGreca, M., Fiorentino, A., Monaco, P., Oriano, P., Temussi, F., 2004. Structure elucidation and phytotoxicity of C13 nor-isoprenoids from Cestrum parqui. Phytochemistry 65, 497–505. Fattorusso, E., Lanzotti, V., Taglialatela-Scafati, O., Di Rosa, M., Ianaro, A., 2000. Cytotoxic saponins from bulbs of Allium porrum L. J. Agric. Food Chem. 48, 3455–3462. Hayes, P.Y., Jahidin, A.H., Lehmann, R., Penman, K., Kitching, W., De Voss, J.J., 2008. Steroidal saponins from the roots of Asparagus racemosus. Phytochemistry 69, 796–804. Hu, K.F., Berenjian, S., Larsson, R., Gullbo, J., Nygren, P., Lovgren, T., Morein, B., 2010. Nanoparticulate Quillaja saponin induces apoptosis in human leukemia cell lines with a high therapeutic index. Int. J. Nanomed. 5, 51–62. Iorizzi, M., Lanzotti, V., Ranalli, G., De Marino, S., Zollo, F., 2002. Antimicrobial furostanol saponins from the seeds of capsicum annuum L. Var. acuminatum. J. Agric. Food Chem. 50, 4310–4316. Judd, W.S., Campbell, C.S., Kellogg, E.A., Stevens, P.F., 1999. Plant Systematics: A Phylogenetic Approach. Sinauer Associates, Sunderland. Lorent, J.H., Quetin-Leclercqb, J., Mingeot-Leclercq, M., 2014. The amphiphilic nature of saponins and their effects on artificial and biological membranes and potential consequences for red blood and cancer cells. Org. Biomol. Chem. 12, 8803–8822. Maisashvili, M.R., Kuchukhidze, D.K., Kikoladze, V.S., Gvazava, L.N., 2012. Steroidal glycosides of gitogenin from Allium rotundum. Chem. Nat. Comp. 48, 86–90. McLennan, M.W., Kelly, W.R., 1984. Cestrum Parqui (Green Cestrum) Poisoning in Cattle. Aust. Vet. J. 61, 289–291.
172
Phytochemistry Letters 22 (2017) 167–173
R.R. Mosad et al.
Sci. 51, 289. Sparg, S.G., Light, M.E., Staden, J.V., 2004. Biological activities and distribution of plant saponins. J. Ethnopharmcol. 94, 219–243. Tschesche, R., Wulff, G., 1963. Uper saponine der spirostanolreihe-IX, die constitution des digitonins. Tetrahedron Lett. 19, 621–634. Yahara, S., Ura, S., Sakamoto, C., Nohara, T., 1994. Steroidal glycosides frorm capsicum annuum. Phytochemistry 37, 831–835.
Mimaki, Y., Yokosuka, A., Sakuma, C., Sakagami, H., Sashid, Y., 2002. Spirostanol pentaglycosides from the underground parts of Polianthes tuberosa. J. Nat. Prod. 65, 1424–1428. Rejinold, N.S., Muthunarayanan, M., Muthuchelianb, K., Chennazhia, K.P., Naira, S.V., Jayakumar, R., 2011. Saponin-loaded chitosan nanoparticles and their cytotoxicity to cancer cell lines in vitro. Carbohydr. Polym. 84, 407–416. Silva, M., Mancinelli, P., Cheul, M., 1962. Chemical study of Cestrum parqui. J. Pharm.
173